Molecular detection and genotype identification of Acanthamoeba species from bronchoalveolar lavage of patients with pulmonary symptoms suspected of cancer.

Acanthamoeba spp. are the most common free-living amoeba worldwide, inducing life-threatening diseases such as Granulomatous Amoebic Encephalitis, pulmonary infection, and amoebic keratitis. This study aimed to identify the FLA and Acanthamoeba genotypes in patients with pulmonary symptoms suspected of cancer in Kashan's hospitals, Kashan, Iran. This cross-sectional study was conducted on 97 bronchoalveolar lavage samples of patients with respiratory symptoms suspected of lung cancer, who were admitted to the Shahid Beheshti Hospital of Kashan from 2019 to 2020. The samples were cultured onto 1.5% non-nutrient agar enriched with killed Escherichia coli and examined for the presence of FLA. Following amoeba isolation and DNA extraction, Acanthamoeba spp. were determined by Polymerase Chain Reaction using JDP1 and JDP2 primers, which amplified a 490 bp fragment from the 18 S rDNA gene. Eighteen Acanthamoeba isolates were sequenced, and the genotypes were identified. The prevalence of FLA and Acanthamoeba and the relationship between symptoms and demographic variables were analyzed with SPSS Software version 16. The prevalence rates of FLA and Acanthamoeba in the BAL samples was 86.6% and 73.2%, respectively. All Acanthamoeba isolates belonged to the T4 genotype. The most symptoms among Acanthamoeba-positive patients were dyspnea and cough; however, their difference was not statistically significant. The findings indicated the high prevalence of FLA and Acanthamoeba in BAL in the population suspected of cancer in Kashan. Since the T4 genotype is a pathogenic genotype of Acanthamoeba, training health and improving sanitation levels would help to prevent infection.

Anti-parasitic effects of resveratrol on protoscolices and hydatid cyst layers.

The main goal of the current study was to evaluate the effectiveness of resveratrol (RESV) on protoscolices and hydatid cysts of Echinococcus granolosus. Echinococcus granolosus protoscolices and hydatid cyst were exposed to RPMI, DMSO, formalin, mebendazole, and different concentrations of RESV in vitro. Then, viability, GGT, and caspase-3 activity of protoscolices were evaluated using light microscopy, colorimetric, and enzymatic assay, respectively. Tissue changes and expression of caspase-3 apoptosis were analyzed on the hydatid cyst wall by histologic and immunohistochemistry methods. The cell toxicity effect of RESV was evaluated on mouse PBMCs by Annexin V-FITC assay. The RESV-treated protoscolices showed loss of viability, increased gamma-glutamyl transpeptidase, and caspase-3 activity with significant differences compared to all control groups (P < 0.05). Dose and time dependence of mortality, GGT, and caspase-3 enzymatic activity was confirmed in the protoscolices of Echinococcus granulosus treated by RESV. Also, the tissue changes and apoptosis were prominent in RESV-treated hydatid cyst layers; however, tissue changes were only time-dependent, and RESV concentration had no apparent effect on tissue. In cell toxicity evaluation, RESV is safe without any significant apoptosis induction from 31.5 to 250 µg/ml; however, it was significant at 350 and 500 µg/ml in PBMCs.

Mobile Phone Radiation: Its Efficacy as Protoscolicidals.

OBJECTIVE: There are different types of radiations, such as microwaves and mobile waves. Certain types of radiofrequency were evaluated in hydatid cyst ablation or as protoscolicidals. This study aimed to assess the influence of mobile waves on hydatid cyst protoscolices. METHODS: Hydatid cysts were collected from the slaughterhouse and transferred to the laboratory. The contents of the cysts were drained in sterile conditions and the protoscolices were rinsed three times with phosphate buffered saline. Equal volumes of protoscolex suspensions were aliquoted into similar tubes. Based on the distance of the samples from the mobile generation waves, the tubes containing the parasitic suspensions were classified into three groups, each of which was further categorised into nine subgroups according to the time of the radiation exposure. The subgroup with zero exposure time was considered the control. RESULTS: It was found that the mortality rate of the protoscolices increases as the distance of the sample from the wave-generation source decreases (p<0.0001). Increasing the time of exposure also improves the mortality rate of protoscolices. CONCLUSION: The mortality rate of protoscolices was directly proportional to the time of exposure and inversely proportional to the distance from the mobile generation waves.

Isolation and Genotyping of Acanthamoeba from Soil Samples in Markazi Province, Iran.

AIM: A previous study confirmed the contamination of water sources with this parasite in Arak, Markazi Province, Iran. The current study investigated soil sources and determined the predominant genotype of Acanthamoeba in this region of Iran. MATERIAL AND METHODS: Forty-eight soil samples, collected from different regions of Arak, Markazi province, Iran, were evaluated in this study. The samples were processed and identified by culturing on a specific medium, performing PCR assay, and sequencing the PCR products. Finally, using the NCBI database, the genotypes were determined. RESULTS: Of 48 soil samples, 33.3% and 31.25% were contaminated with Acanthamoeba according to the culture and molecular assays, respectively. The majority of these isolates belonged to the T4, T5 and T6 genotypes of Acanthamoeba. CONCLUSION: The genotypes of most isolates from soil samples in Arak similar to other regions of Iran belong to T4 genotype of this parasite. New sequence accession numbers include MG066681 and MG298785-MG298794.

Toxoplasmosis: Seroprevalence in pregnant women, and serological and molecular screening in neonatal umbilical cord blood.

Toxoplasmosis is a common zoonotic disease that can also be transmitted from the mother to the embryo, with the risk of congenital infection varying around the world. The aim of this study was to screen pregnant women and their neonates for toxoplasmosis by serologic and molecular methods and assess the impact of risk factors associated with toxoplasmosis on the rate of congenital infection. This study was conducted at a regional maternity hospital in Arak, the capital of the Markazi Province in Iran, during a period of six months. All selected pregnant women (n=261) and the corresponding cord blood samples were serologically screened for toxoplasmosis, with seropositive samples also undergoing molecular testing. Demographic data, as well as information related to the risk factors associated with the transmission of the disease, were collected from mothers and their neonates. The detection of anti-Toxoplasma antibodies and the extraction of DNA from blood samples were conducted using commercial kits. Results showed that the sera of 87 maternal blood samples (33.3%) and 40 cord blood samples (15.3%) were positive for anti-Toxoplasma antibodies (IgG and/or IgM). Molecular screening of the seropositive samples only identified one positive cord blood sample. In other words, the diagnosis of congenital toxoplasmosis was definitive in only one neonate. There was no significant association between the risk of parasite transmission and neonatal seropositivity (p >0.05). Therefore, the results showed that the prevalence of congenital toxoplasmosis in the studied area was consistent with the global rate and suggest that the implementation of newborn screening and follow-up testing could help reduce the disease risk.

Detection of Specific Antibody Reactivity to Toxocara Larval Excretory-secretory Antigens in Asthmatic Patients (5-15 Years).

BACKGROUND & PURPOSE: Humans act as an intermediate host for Toxocara canis and Toxocara cati. Toxocara may be an important risk factor for asthma in humans. The aim of the present study was to evaluate immunoglobulin G (IgG) anti-Toxocara canis antibody, using enzyme-linked immunosorbent assay (ELISA) in asthmatic patients (aged 5-15 years), referring to a clinic of pulmonary diseases in Arak, Iran. MATERIALS & METHODS: In this bi-group cross sectional study, serum samples were collected from 110 children with confirmed asthma and 70 children without asthma within one year. IgG anti-Toxocara antibody was detected viaELISA method. The collected data were analyzed, using SPSS. RESULTS: The seroprevalence of antibodies against Toxocara species was estimated at 1.8% (two males) in asmathic children viaELISA method; however, no antibodies against Toxocara canis were detected in the control group. There was no significant correlation between the frequency of antibodies against Toxocara and variables such as age, gender, or place of residence (P>0.05). Moreover, the frequency of antibodies against Toxocara was not significantly correlated with contact with dogs, consumption of unwashed fruits and vegetables, or use of raw/undercooked sheep liver (P>0.05). CONCLUSION: The present study showed anti-Toxocara antibody in 1.8% of asthmatic children and determined the seroprevalence of toxocariasis in asthmatic children and adolescents in Arak, Iran. Based on the findings, the low rate of infection with Toxocara among asthmatic children may be attributed to acceptable personal hygiene and religious considerations.

An In Vitro Evaluation of Ozonized Organic Extra-Virgin Olive Oil on Giardia Lamblia Cysts.

BACKGROUND: Giardia lamblia is a common intestinal parasite that has been reported all over the world. OBJECTIVES: This study was conducted to evaluate the effect of ozonized organic extra-virgin olive oil on the cyst of G. lamblia. METHODS: The olive oil was ozonized based on international standards and confirmed by the world health organization (WHO) at various times in a generator. The ozone concentration of olive oil was adjusted at 32, 64, 96, 128, 160 mg/g based on ozone absorption. Giardia lamblia cysts were isolated from heavily infected stool samples and the sucrose gradient flotation technique. Five groups of triple tubes containing Giardia cysts were exposed to olive oil with 32, 64, 96, 128, 160 ozone concentrations, and the sixth and seventh groups were exposed to non-ozonized olive oil and normal saline, respectively. The tubes were placed at room temperature, and every four hours, the mortality of the Giardia cysts was assessed. RESULTS: The results showed that the first five groups' mortality rate of Giardia cysts reached 100% in 100 hours. An increasing concentration of ozone in olive oil leads to an increase in the mortality rate of Giardia cysts. The results showed a significant difference in the mean time of the mortality in all the groups (P ≤ 0.05). Furthermore, the higher fatality effect of ozonized organic extra-virgin olive oil (Ozonized Olive Oil = OZO) was proved in comparison with metronidazole in vitro. CONCLUSIONS: We concluded that ozonized organic extra-virgin olive oil was a growth inhibitor of Giardia cysts, and concerning its compatibility with a biological system, it is recommended for further clinical trials.

Histological Changes of the Ovary in Pregnant Mice Vaginally Exposed to Toxoplasma gondii.

BACKGROUND: Congenital toxoplasmosis is one cause of abortion. Infection can disrupt ovarian cycles and because toxoplasmosis is an infectious disease may have a similar effect on the ovaries. The purpose of this study was to investigate the pathological changes in the ovaries due to toxoplasmosis. METHODS: Tachyzoites of Toxoplasma gondii were harvested from peritoneal fluid of mice, experimentally infected. Two females and one male mouse were housed per cage for mating in the overnight. The pregnant mice were divided into experimental and control groups. Experimental group were infected by parasite but the control group received the normal saline. The experimental and control mice were euthanized. Ovaries and uterine horns of animals were removed and prepared for light microscopy. RESULTS: Ovaries of infected pregnant mice presented gross morphological differences compared to the control groups. In ovaries of experimental groups, changes of corpus luteum were observed. The comparison of experimental and control groups revealed that the number of primary follicles, secondary follicle, atretic primary follicles and atretic secondary follicles had significant differences (P≤0.001). CONCLUSION: Toxoplasma gondii alters ovarian follicular growth and development in mice. In addition, it alters number of different phases of follicles and corpus luteum in ovaries of mice.

Identification of Malassezia Species Isolated from Patients with Pityriasis Versicolor Using PCR-RFLP Method in Markazi Province, Central Iran.

BACKGROUND: The lipophilic yeasts of Malassezia species are members of the normal skin microbial that are cause of pityriasis versicolor. Pityriasis versicolor is a common superficial fungal infection with world-wide distribution. The phenotypic methods for identification of Malassezia species usually are time consuming and unreliable to differentiate newly identified species. But DNA-based techniques rapidly and accurately identified Malassezia species. The purpose of this study was isolation and identification of Malassezia Species from patients with pityriasis versicolor by molecular methods in Markazi Province, Central Iran in 2012. METHODS: Mycologic examinations including direct microscopy and culture were performed on clinical samples. DNA extraction was performed from colonies. The ITS1 region of rDNA from isolates of Malassezia species were amplified by PCR reaction. The PCR were digested by Cfo I enzyme. RESULTS: From 70 skin samples, were microscopically positive for Malassezia elements, 60 samples were grown on culture medium (85.7%). Using PCR-RFLP method, that was performed on 60 isolates, 37(61.6%) M. globosa, 14(23.3%) M. furfur, 5(8.4%) M. sympodialis and 4(6.7%) M. restrictawere identified. In one case was isolated M. globosa along with M. restricta. CONCLUSION: The PCR-RFLP method is a useful and reliable technique for identification of differentiation of Malas-sezia species.

Effects of Toxoplasma gondii Infection in Level of Serum Testosterone in Males with Chronic Toxoplasmosis.

BACKGROUND: Toxoplasma gondii is an intracellular protozoan parasite that infects human and animals. Toxoplasma parasites are isolated from different parts of animals even from semen but there are little information about the effect of toxoplasmosis on fertility in animals and humans. In present study, the effect of chronic toxoplasmosis on serum levels of testosterone in men was studied. METHODS: In this case-control study, 1026 men referred to Arak Post Marriage Center were selected. Three ml of blood samples were collected and sera separated by centrifugation at room temperature. These sera were analyzed for detection of anti-T. gondii IgG antibody. Next 365 positive sera were selected as cases and also the same number of negative sera (365) as controls. Finally the level of testosterone was analyzed for the cases and controls samples. RESULT: Serological tests on the sera of 1,026 men in Arak City showed that 365 of them had anti-Toxoplasma antibody. Comparison of testosterone concentration in case and control groups showed that testosterone concentration in case group was less than control group and this difference was statistically significant (P<0.05). CONCLUSION: The chronic toxoplasmosis could affect reproductive parameters in men.

Toxoplasmosis: Experimental Vaginal Infection in NMRI Mice and Its Effect on Uterin, Placenta and Fetus Tissues.

BACKGROUND: Toxoplasma gondii is an important zoonotic pathogen. Vertical transmission of the parasite occurs when females were infected primarily during gestation. This parasite is transmitted to the fetus through the placenta and may cause miscarriage, permanent neurological damage, premature birth and visual impairment. It has been found that mouse is susceptible to Toxoplasma and is particularly an interesting model to the study of congenital infection but whether the entry of T. gondii through vagina route is involved in transmission of the parasite to the placenta and fetus or not. OBJECTIVES: The current study aimed to find a route of infection which perhaps carried the parasite under natural conditions in human. MATERIALS AND METHODS: In the current experimental study, two 6-8 week NMRI female mice were crossed with one male. The pregnant mice were divided into 2 groups: experimental group that was infected by parasite via intra-vaginal (IV) and control group that received the same volume of normal saline via IV. One mouse from each group was killed on the fifth day after infection. The peritoneal fluid, ovary and uterus of mouse samples were taken and divided into two parts. One part used for DNA extraction and the other was kept in formalin and sent for histological study. These steps were repeated seven times and at least 10 mice in each group (case and control) were studied by molecular and histological methods. RESULTS: PCR using DNA extracted from the experimental group showed that the parasite existed in tissues of the uterus and placenta but not in the embryos and peritoneal fluid. PCR using DNA extracted from the control group was negative. CONCLUSIONS: Tachyzoite of Toxoplasma and DNA of this parasite were observed in sub mucosa and muscles of the uterus and in the villis of placenta, but not in histological sections of the fetus. Therefore, histological and molecular results were consistent.